CN113425671A - 一种调控肿瘤微环境免疫凝胶的制备方法及其应用 - Google Patents
一种调控肿瘤微环境免疫凝胶的制备方法及其应用 Download PDFInfo
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Abstract
一种调控肿瘤微环境免疫凝胶的制备方法,包含以下步骤:(1)合成掺杂NO的普鲁士蓝纳米粒子;(2)对普鲁士蓝纳米粒子进行氨基化修饰;(3)将Anti‑SIRPα修饰于普鲁士蓝纳米粒子表面;(4)将CSF‑1R小分子抑制剂BLZ945负载于普鲁士蓝粒子的孔穴中;(5)制备胶凝制剂与凝血酶溶液;(6)将步骤(4)得到的负载BLZ945的普鲁士蓝纳米粒子与胶凝制剂溶液混合均匀,加入凝血酶溶液,在凝血酶的刺激下即得免疫凝胶。本发明制备方法简单、材料易得,效果优异,以普鲁士蓝作为载体,可实现对药物的高效负载;制备的免疫凝胶对癌症术后复发进行治疗,既保证了局部免疫治疗纳米粒子的持续控制释放,又实现了术后创口的修复与愈合,有显著的社会和经济效益。
Description
技术领域
本发明涉及纳米生物医药领域,特别是一种调控肿瘤微环境免疫凝胶的制备方法及其应用。
背景技术
肿瘤,特别是恶性肿瘤已是常见多发病,严重危害人的身心健康和生命,目前治疗肿瘤有多种途径,主要是手术加药物控制,不可否认的是,对于瘤体明确、局部和较小的实体瘤来说,手术切除可实现生存率的提高。但癌症术后居高不下的复发率严重影响患者手术疗效,是导致治疗失败的主要原因。诸多动物和临床试验数据表明癌症的术后复发、转移是一个多步骤、多环节、多因素和多分子参与的复杂过程,涉及机体、微环境和肿瘤组织三个方面因素。因此,免疫疗法逐渐进入了人们的视野,调动起机体自身的免疫系统去识别并杀死肿瘤细胞,是未来最有可能根治肿瘤的治疗途径。
肿瘤相关巨噬细胞(Tumor-associated macrophage,TAM)是肿瘤微环境的重要组成成分。作为肿瘤免疫网络关键节点成员,TAM不但参加固有免疫,而且调节获得性免疫,调控肿瘤的生长、转移及耐药,甚至影响着免疫治疗的成败。TAM表型具有高度可塑性,可在M1与M2两个亚型之间转换,微环境中TAM两个亚型处于动态平衡状态。M1亚型TAM可以激活肿瘤免疫应答,分泌抗肿瘤细胞因子(如TNF-α、IL-12等);M2亚型TAM则抑制肿瘤免疫应答,分泌促肿瘤细胞因子(如TGF-β、IL-10等)。诸多研究已表明M2亚型TAM在肿瘤部位的数量与预后效果呈负相关,故而靶向TAM的肿瘤微环境调控成为极具应用前景的免疫疗法。
靶向TAMs的治疗法则(策略)主要分为三个部分:(1)抑制TAM的招募;(2)靶向杀伤TAM;(3)重塑TAM的功能。为了逆转由M2亚型TAM导致的肿瘤免疫抑制性微环境,采用重塑TAM的策略。目前,调节TAM极化的主要技术有低剂量放疗、Anti-CD40抗体激动剂、STAT3抑制剂、CSF-1R抑制剂、NF-κB抑制剂等。
硝普钠是治疗高血压急症和急性左心衰竭的常用药物,可通过血管平滑肌代谢产生一氧化氮(NO)而发挥其强大的血管舒张作用。随着人们对NO了解的不断深入,研究者发现NO在肿瘤微环境中发挥着重要的作用。NO一方面可有效改善肿瘤微环境中的缺氧、血液灌注不足等缺陷,促进TAM向M1型极化,激活T细胞发挥免疫效应;另一方面NO还可以通过下调缺氧诱导因子,降低肿瘤细胞表面PD-L1蛋白的表达,促进T细胞发挥免疫作用。
MCSF-CSF-1R信号轴阻断是针对TAM及其促肿瘤作用的高特异性治疗。癌细胞所分泌的集落刺激因子(Macrophage colony stimulating factor,MCSF)与巨噬细胞表面表达的集落刺激因子1受体(Colony stimulating factor 1 receptor,CSF1-R)结合后,激活相关下游通路,导致TAM发生M2亚型的极化,故而MCSF-CSF-1R信号轴对巨噬细胞的分化和存活至关重要,其过度表达通常与不良预后有关。常用阻断MCSF-CSF-1R的小分子抑制剂有ARRY-382、PLX7486、PLX3397、BLZ945以及JNJ-40346527,单克隆抗体有Emactuzumab、AMG820、IMC-CS4等。临床已采用通过靶向MCSF-CSF-1R信号轴对恶性胶质瘤、乳腺癌和子宫颈癌等类实体肿瘤进行治疗,单一抑制剂的治疗效果疗效甚微。
CD47-SIRPα信号轴是另一个可用免疫治疗的靶点,信号调节蛋白α(Signalregulatory proteins α,SIRPα)是一种通过细胞质区域与酪氨酸磷酸酶SHP-1和SHP-2结合的跨膜蛋白,主要表达于神经元、树突状细胞和巨噬细胞表面。肿瘤细胞表面的CD47蛋白与巨噬细胞表面的SIRPα结合后,会使SIRPα胞质区内的ITIMs磷酸化,Src同源磷酸酶、SHP-1和SHP-2会被募集至巨噬细胞内,而这些磷酸酶反过来又抑制肌球蛋白Ⅱ在吞噬突触的积累,抑制巨噬细胞的吞噬作用。CD47-SIRPα信号轴的阻断可增强巨噬细胞对肿瘤成分的摄取,改善抗原递呈及促吞噬作用共刺激信号的产生,还导致更多促炎性免疫反应的发生。新型CD47拮抗剂(Hu5F9-G4,CC-9000)和SIRPα阻断性单抗(TT1-621,ALX148)目前正在进行I期临床试验。
纤维蛋白单体在凝血酶及Ca2+等催化因子作用下,可聚合形成凝胶类生物大分子材料。纤维蛋白凝胶由于其优异的加速伤口愈合能力,室温下稳定以及同时适用于凝血功能障碍的患者而具有广泛的应用前景。并且作为一种生物可降解材料,已被FDA批准生产应用。除此之外,与水凝胶相比,纤维蛋白凝胶具有更加优异的机械强度和孔隙率,以纤维蛋白凝胶为输送载体,可以实现纳米粒子的缓慢持续释放,提高生物利用度,充分发挥药物的作用,但至今未见公开报道。
发明内容
针对上述情况,为克服现有技术之缺陷,本发明之目的就是提供一种调控肿瘤微环境免疫凝胶的制备方法及其应用,可有效解决目前免疫治疗药物所存在的生物利用度低、单一抑制剂作用效果不理想的问题。
为实现上述目的,本发明解决的技术方案是,一种调控肿瘤微环境免疫凝胶的制备方法,包含以下步骤:
(1)合成掺杂NO的普鲁士蓝纳米粒子;
(2)对普鲁士蓝纳米粒子进行氨基化修饰,得氨基化修饰的掺杂NO的普鲁士蓝纳米粒子;
(3)将Anti-SIRPα 修饰于普鲁士蓝纳米粒子表面,得Anti-SIRPα 修饰的普鲁士蓝纳米粒子;
(4)将CSF-1R小分子抑制剂BLZ945负载于普鲁士蓝粒子的孔穴中,得负载BLZ945的普鲁士蓝纳米粒子;
(5)制备胶凝制剂与凝血酶溶液;
所述的胶凝制剂为壳聚糖及其衍生物、透明质酸及其衍生物、藻酸及其衍生物、纤维蛋白及其衍生物或聚链烯基聚醚交联的丙烯酸;
(6)将步骤(4)得到的负载BLZ945的普鲁士蓝纳米粒子与胶凝制剂溶液混合均匀,加入凝血酶溶液,在凝血酶的刺激下即得免疫凝胶。
所述方法制备的免疫凝胶在制备防止癌症术后复发药物中的应用。
本发明制备方法简单、材料易得,效果优异,以普鲁士蓝作为载体,可实现对药物的高效负载;此外,普鲁士蓝本身具有过氧化氢酶活性,可分解机体内的过氧化氢产生氧气,缓解免疫抑制性微环境中的乏氧状态,制备的免疫凝胶对癌症术后复发进行治疗,既保证了局部免疫治疗纳米粒子的持续控制释放,又实现了术后创口的修复与愈合,相比于系统性的免疫药物,该免疫凝胶采用的局部性免疫治疗毒性更低,更经济便利,有显著的社会和经济效益。
附图说明
图1是本发明实验1制备的发挥免疫效应纳米粒子(BLZ945@NP/Anti-SIRPα NPs)的TEM图;
图2为本发明实验2制备的免疫凝胶(BLZ945@NP/Anti-SIRPα Gel)的SEM图;
图3为本发明实验1中制备的发挥免疫效应纳米粒子(BLZ945@NP/Anti-SIRPαNPs)的SDS-PAGE凝胶电泳图;
图4为本发明实验1制备的掺杂NO的普鲁士蓝纳米颗粒(NP NPs)、表面氨化的普鲁士蓝纳米颗粒(NP-NH2 NPs)、负载BLZ945及Anti-SIRPα 修饰的普鲁士蓝纳米颗粒(BLZ945@NP/Anti-SIRPα NPs)粒径、电位分布图;
图5为本发明BLZ945@NP/Anti-SIRPα NPs在体外作用效果评价流式结果图;
图6为本发明制剂的安全性评价图。其中a为小鼠的体重变化曲线图,b为小鼠各组织的病理切片结果图。
具体实施方式
以下结合附图和具体情况对本发明的具体实施方式作详细说明。
本发明在具体实施中,可由以下实施例给出。
实施例1
本发明一种调控肿瘤微环境免疫凝胶的制备方法,包含以下步骤:
(1)、合成掺杂NO的普鲁士蓝纳米粒子:称取PVP-K30粉末1.2 g,溶于30 mM的K3[Fe(CN)6]溶液中,同时加入等摩尔质量的硝普钠,用浓度12 M的HCl调节PH 为 0,室温下搅拌至溶液变为澄透明的黄色,在80℃条件下避光搅拌12 h,用离心洗涤溶剂对产物进行洗涤离心3次,10000 rpm/min条件下离心30 min,随后置于真空干燥箱中干燥12 h,即得到粒径为50 nm的掺杂NO的普鲁士蓝纳米粒子;
所述的离心洗涤溶剂为丙酮、乙醇、超纯水中的一种或按体积任意比混合的两种;
(2)、氨基化修饰:取掺杂NO的普鲁士蓝纳米粒子10 mg与2 mg分子量为1800的氨基化组分置于40 mL超纯水中,磁力搅拌24 h后,置于透析袋中透析24 h,除去未修饰上的、游离的氨基化组分,用超纯水洗涤离心,10000 rpm条件下离心10 min,即得氨基化修饰的普鲁士蓝纳米粒子;
所述的氨基化组分为聚醚酰亚胺、聚丙烯胺基盐酸盐、聚酰胺-胺型中的一种或按体积任意比混合的两种;
(3)、将Anti-SIRPα 修饰于普鲁士蓝纳米粒子表面:取氨基化修饰的普鲁士蓝纳米粒子10 mg,加入新鲜配制的活化试剂2 mg/mL的EDC与10 mg/mLNHS溶液,混合均匀后置于25℃摇床中活化1 h,将活化后的产物以9000 rpm离心10 min洗涤3-5次,以除去游离的EDC和NHS分子,得氨基活化的普鲁士蓝纳米粒子,并产物分散于PBS溶液中。加10 μg的SIRPα 抗体至氨基活化的普鲁士蓝纳米粒子中,用移液器轻轻吹打混匀,在4℃条件下孵育过夜,采用6000 rpm转速离心,沉淀分散于PBS溶液中,得SIRPα 抗体修饰的普鲁士蓝纳米粒子;
(4)、负载BLZ945:取经抗体修饰的普鲁士蓝纳米粒子10 mg超声分散于超纯水中,得第一溶液,取10 mg的BLZ945小分子抑制剂溶于DMSO溶液中,得第二溶液,将第二溶液在室温搅拌下加入到第一溶液中,搅拌反应24 h后,置于透析袋中透析24 h,去除游离的BLZ945,用超纯水洗涤离心,10000 rpm条件下离心10 min,得负载BLZ945的普鲁士蓝纳米粒子;
所述负载BLZ945的普鲁士蓝纳米粒子为注射剂、口服剂或植入剂,优选为植入剂,将制剂制成凝胶或支架形式,可用于术后复发的治疗,
(5)、制备胶凝制剂与凝血酶溶液:将胶凝制剂置于生理盐水溶液中,37℃条件下搅拌10 min,配置终浓度为10 mg/mL的胶凝制剂溶液,将冻干凝血酶置于40 mM氯化钙溶液中,配置终浓度为50 U/mL的凝血酶溶液;
(6)、制得免疫凝胶:取步骤(4)中得到的负载BLZ945的普鲁士蓝纳米粒子5 mg,将其分散至10 mg/mL的胶凝制剂溶液中,然后取等体积的凝血酶溶液与之混合,置于37℃恒温箱中孵育1-3 min,即得免疫凝胶;
所述的免疫凝胶为喷雾、注射溶液或涂抹凝胶,优选喷雾形式。
实施例2
一种调控肿瘤微环境免疫凝胶的制备方法,包含以下步骤:
(1)、合成掺杂NO的普鲁士蓝纳米粒子:称取PVP-K30粉末1.2 g,溶于30 mM的K3[Fe(CN)6]溶液中,同时加入等摩尔质量的硝普钠,用浓度12 M的HCl调节PH 为 0,室温下搅拌至溶液变为澄透明的黄色,在80℃条件下避光搅拌12 h,分别用乙醇、乙醇与超纯水体积比 1:1混合液和丙酮,依次对产物进行洗涤、离心,10000 rpm/min条件下离心30 min,随后置于真空干燥箱中干燥12 h,即得到粒径为50 nm的掺杂NO的普鲁士蓝纳米粒子;
(2)、氨基化修饰:取掺杂NO的普鲁士蓝纳米粒子10 mg与2 mg分子量为1800的聚丙烯胺基盐酸盐置于40 mL超纯水中,磁力搅拌24 h后,置于透析袋(截留分子量8 kDa)中透析24 h,除去未修饰上的、游离的聚丙烯胺基盐酸盐,用超纯水洗涤离心,10000 rpm条件下离心10 min,即得氨基化修饰的普鲁士蓝纳米粒子;
(3)、将Anti-SIRPα 修饰于普鲁士蓝纳米粒子表面:取氨基化修饰的普鲁士蓝纳米粒子10 mg,加入新鲜配制的活化试剂2 mg/mL的EDC与10 mg/mLNHS溶液,混合均匀后置于25℃摇床中活化1 h,将活化后的产物以9000 rpm离心10 min洗涤3次,以除去游离的EDC和NHS分子,得氨基活化的普鲁士蓝纳米粒子,并产物分散于PBS溶液中。加10 μg的SIRPα抗体至氨基活化的普鲁士蓝纳米粒子中,用移液器轻轻吹打混匀,在4℃条件下孵育过夜,采用6000 rpm转速离心,沉淀分散于PBS溶液中,即得粒径为40-100 nm的Anti-SIRPα 修饰的普鲁士蓝纳米粒子;
(4)、负载BLZ945:取经抗体修饰的普鲁士蓝纳米粒子10 mg超声分散于超纯水中,得第一溶液,取10 mg的BLZ945小分子抑制剂溶于DMSO溶液中,得第二溶液,将第二溶液在室温搅拌下加入到第一溶液中,搅拌反应24 h后,置于透析袋(截留分子量8 kDa)中透析24h,去除游离的BLZ945,用超纯水洗涤离心后,10000 rpm条件下离心10 min,即得负载BLZ945的普鲁士蓝纳米粒子;
(5)、制备纤维蛋白原溶液与凝血酶溶液:将冻干纤维蛋白原置于生理盐水溶液中,37℃条件下搅拌10 min,配置终浓度为10 mg/mL的纤维蛋白原溶液,将冻干凝血酶置于40 mM氯化钙溶液中,配置终浓度为50 U/mL的凝血酶溶液;
(6)、制得免疫凝胶:取步骤(4)中得到的负载BLZ945的普鲁士蓝纳米粒子5 mg,将其分散至10 mg/mL的纤维蛋白原溶液中,然后取等体积的凝血酶溶液与之混合,置于37℃恒温箱中孵育1-3 min,即得免疫凝胶。
实施例3
一种调控肿瘤微环境免疫凝胶的制备方法,包含以下步骤:
(1)、合成掺杂NO的普鲁士蓝纳米粒子:称取PVP-K30粉末1.2 g,溶于30 mM的K3[Fe(CN)6]溶液中,同时加入等摩尔质量的硝普钠,用浓度12 M的HCl调节PH = 0,室温下搅拌至溶液变为澄透明的黄色。随后在80℃条件下避光搅拌12 h,分别用2.5倍体积的乙醇、乙醇与超纯水体积比 1:1混合液和超纯水对产物进行洗涤离心(10000 rpm/min,30 min),置于真空干燥箱中40℃干燥12 h,即得到粒径为50 nm的掺杂NO的普鲁士蓝纳米粒子(即NPNPs);
(2)、氨基化修饰:取NP NPs 10 mg与2 mg分子量为1800的聚醚酰亚胺置于40 mL超纯水中,磁力搅拌24 h后,置于透析袋(截留分子量8 kDa)中透析24 h,除去未修饰上的、游离的聚醚酰亚胺,用超纯水洗涤离心(10000 rpm,10 min),即得氨基化修饰的普鲁士蓝纳米粒子(即NP-NH2 NPs);
(3)、将Anti-SIRPα 修饰于普鲁士蓝纳米粒子表面:取NP-NH2 NPs 10 mg,加入新鲜配制的活化试剂2 mg/mL的EDC与10 mg/mLNHS溶液,混合均匀后置于25℃摇床中活化1h,将活化后的产物以9000 rpm离心10 min洗涤4次,以除去游离的EDC和NHS分子,得氨基活化的普鲁士蓝纳米粒子,并将产物分散于PBS溶液中。加10 μg的SIRPα 抗体至氨基活化的普鲁士蓝纳米粒子中,用移液器轻轻吹打混匀,在4℃条件下孵育过夜,采用6000 rpm转速离心,沉淀分散于PBS溶液中,即得粒径为40-100 nm的Anti-SIRPα 修饰的普鲁士蓝纳米粒子(即NP/Anti-SIRPα NPs);
(4)、负载BLZ945:取NP/Anti-SIRPα NPs 10 mg超声分散于超纯水中,得第一溶液,取10 mg的BLZ945溶于15 ml的DMSO溶液中,得第二溶液,将第二溶液在室温搅拌下加入第一溶液中,搅拌反应24 h后,置于透析袋(截留分子量8 kDa)中透析24 h,去除游离的BLZ945,用超纯水洗涤离心(10000 rpm,10 min),即得负载BLZ945的普鲁士蓝纳米粒子(即BLZ945@NP/Anti-SIRPα NPs );
(5)、制备纤维蛋白原溶液与凝血酶溶液:将冻干纤维蛋白原置于生理盐水溶液中,37℃条件下搅拌10 min,配置为终浓度为10 mg/mL的纤维蛋白原溶液。将冻干凝血酶置于40 mM氯化钙溶液中,配置成终浓度为50 U/mL的凝血酶溶液;
(6)、制得免疫凝胶:取BLZ945@NP/Anti-SIRPα NPs 5 mg,将其分散至10 mg/mL的纤维蛋白原溶液中,然后取等体积的凝血酶溶液与之混合,置于37℃恒温箱中孵育1-3min,即得免疫凝胶(即BLZ945@NP/Anti-SIRPα Gel)。
本发明一种调控肿瘤微环境免疫凝胶的制备方法及其应用,用于预防癌症术后复发的治疗,取得了很好的效果,以实例3为例,相关试验资料如下:
实验1
步骤(4)制备的Anti-SIRPα 修饰的普鲁士蓝纳米粒子(BLZ945@NP/Anti-SIRPαNPs)的透射电镜图(TEM),见附图1,从图中可见,BLZ945@NP/Anti-SIRPα NPs呈类球形,外层包覆一层抗体,其均一性良好,粒径约为70 nm左右。
实验2
步骤(6)最终制得的免疫凝胶BLZ945@NP/Anti-SIRPα Gel的扫描电镜图(SEM),见附图2,从图中可见,纳米粒子成功嵌合在纤维蛋白凝胶的孔隙中,该凝胶内部孔洞均匀,结构致密,呈现多孔纤维状网络结构。
实验3
SDS-PAGE凝胶电泳
1)样品的制备
取BLZ945@NP/Anti-SIRPα NPs 50 mg,加入3-5 mL的超纯水搅拌使之溶解,4000rpm离心15 min后,取上清液加入等体积的20%三氟乙酸聚沉,离心,弃去上清,沉淀用70%的丙酮溶液洗涤3-5次,挥干丙酮后,加入适量PBS溶解沉淀,该溶液可用于后续电泳实验;然后取适量的BLZ945@NP/Anti-SIRPα NPs离心洗涤的上清以及SIRPα 抗体,同样的方法处理,制备出用于后续电泳实验的样品溶液;
将样品与5X样品缓冲液在一个Ep管中混合均匀后,放入100℃水浴中煮沸5-10min,去掉亚稳态聚合,冷却后4000 rpm离心4 min,取上清点样;
2)分离胶及浓缩胶的制备
分离胶的制备:
将玻璃板、样品梳、Spacer用洗涤剂洗净,用超纯水冲洗数次,再用乙醇擦拭,晾干,将两块洗净的玻璃板之间加入Spacer,按照Bio-Rad MiniⅡ/Ⅲ说明书提示装好玻璃板,配制10%分离胶(公知技术),向玻璃板间灌制分离胶,丙三醇液封,18-22 min后胶即可聚合;
浓缩胶的制备:
配制6%浓缩胶(公知技术),混匀;弃去丙三醇,吸干表面液体后灌制浓缩胶,随后将样品梳插入;
3)上样
浓缩胶凝结后,向上槽内倒入电泳缓冲液,拔出样品梳,向每孔内加入10 μL样品;
4)跑胶
加样完成后,在电泳槽内加入电泳缓冲液,连接好泠凝水,接通电源,进行电泳,稳压90 V,电泳90 min,进入分离胶后,电压改为120 V,待溴酚蓝距凝胶边缘约5 mm时,停止电泳;
5)凝胶板剥离与固定
电泳结束后,取开玻璃板,将凝胶板做好标记后放在大培养皿内,用蒸馏水洗涤1-3次,加入固定液进行固定40-60 min,倒出固定液,蒸馏水清洗3-5次;
6)染色
固定清洗结束后,倒入染色液,染色40-60 min,染色液中已看不到凝胶本身颜色表明染色充分;
7)脱色
染色结束后,蒸馏水清洗3-5次,倒入脱色液进行脱色,确保脱色液可充分覆盖凝胶,脱色至背景基本上被脱去、蛋白条带清晰即可。
图3为BLZ945@NP/Anti-SIRPα NPs的SDS-PAGE凝胶电泳图。1号泳道表示BLZ945@NP/Anti-SIRPα NPs离心洗涤的上清,2号泳道表示SIRPα 抗体,3号泳道表示BLZ945@NP/Anti-SIRPα NPs,从图中可见表面Anti-SIRPα 修饰普鲁士蓝纳米粒子(BLZ945@NP/Anti-SIRPα NPs)与SIRPα 抗体的蛋白条带相一致,但离心洗涤的上清则无条带显示,说明Anti-SIRPα 已经成功修饰于普鲁士蓝纳米粒子表面。
实验4
分别取掺杂NO的普鲁士蓝纳米颗粒(NP NPs)、表面氨化的普鲁士蓝纳米颗粒(NP-NH2 NPs)以及Anti-SIRPα 修饰的普鲁士蓝纳米颗粒(BLZ945@NP/Anti-SIRPα NPs)样品分别置于2mL的超纯水中,涡旋使之分散均匀,然后取500 μL的样品,分别置于方形粒径杯与U型电位杯中,借助马尔文激光粒度仪(Zetasizer Nanosize,Nano ZS90)测量各制剂的粒径分布和表面电势变化;
取经修饰后的普鲁士蓝纳米颗粒(BLZ945@NP/Anti-SIRPα NPs)样品分散于超纯水中置于4℃、25℃条件下放至48 h,记录其水合粒径的变化;
另取一份经修饰后的普鲁士蓝纳米颗粒(BLZ945@NP/Anti-SIRPα NPs)样品分散于血清培养基中置于4℃、25℃条件下放至48 h,记录其水合粒径的变化。
图4为掺杂NO的普鲁士蓝纳米颗粒(NP NPs)、表面氨化的普鲁士蓝纳米颗粒(NP-NH2 NPs)以及Anti-SIRPα 修饰的普鲁士蓝纳米颗粒(BLZ945@NP/Anti-SIRPα NPs)的粒径、电位分布图,从图中可见,经修饰后粒经发生变化,电位变化由负到正再到负。这是因为掺杂NO的普鲁士蓝纳米颗粒带负电,聚醚酰亚胺呈现较强的正电性,故而氨基化修饰后,NP-NH2 NPs带正电性,Anti-SIRPα 修饰后BLZ945@NP/Anti-SIRPα NPs电位发生反转,图4中电位及粒径的变化也表明成功制备了BLZ945@NP/Anti-SIRPα NPs。
实验5
体外作用效果评价-TAM表型逆转实验
1)取处于对数生长期的RAW264.7巨噬细胞用完全培养基配制成均匀的细胞悬浮液,然后接种于12孔板中,细胞密度为1 x 105个/孔,置于5% CO2、37℃的细胞培养箱条件下培养24 h;
2)将上清移去,分别在培养基中加入1 μg/mL的脂多糖或40 ng/mL的IL-4,然后置于培养箱中孵育24 h以诱导M1、M2型TAM的产生,空白组加入正常培养基,所得巨噬细胞为M0型;
3)取上述诱导好的巨噬细胞,用细胞刮刀轻轻刮下后,用Ep管收集,用PBS缓冲液清洗3次后,加入含有1% BSA的缓冲液封闭1 h,离心,弃上清,加入200 μL的抗体在冰上孵育1 h,用流式细胞仪检测,以证明成功诱导了M1型、M2型TAM的产生;
4)取上述经IL-4诱导的M2型巨噬细胞,弃去原培养基,分别加入含NP NPs、BLZ945@NP NPs、NP/Anti-SIRPα NPs以及BLZ945@NP/Anti-SIRPα NPs的培养基,置于培养箱中孵育4 h后,弃去原培养液,加入完全培养基继续培养24 h后,弃去废旧培养基,PBS缓冲液清洗3次后加入1 mL浓度为20 μg/mL的CD86/PE、CD206/FITC培养基继续孵育2 h,消化收集细胞,置于流式细胞仪上检测。
图5为BLZ945@NP/Anti-SIRPα NPs在体外作用效果评价,如图流式结果显示,NPNPs、BLZ945@NP NPs以及NP/Anti-SIRPα NPs均表现出对M2型巨噬细胞一定的逆转作用,但其作用效果有限,BLZ945@NP/Anti-SIRPα NPs可发挥的逆转作用更为强大,逆转效果最强。故而BLZ945和Anti-SIRPα 的联合使用,可发挥协同作用的功效,更大程度地逆转M2型巨噬细胞表型,进而实现对肿瘤免疫抑制性微环境的调控。
实验6
取对数生长期的H22肝癌细胞,以2 x 106/mL的细胞密度对小鼠进行接种。选取正常6-8周龄的BALB/c雌性小鼠,将小鼠右侧肋系的毛发脱去,用75%酒精棉球消毒,以每只100 μL的细胞数量皮下接种于小鼠的右侧肋系皮下腹部处。待肿瘤体积达到100 mm3左右时,对其进行分组,每组6只。具体分组为(1)NS;(2)NP NPs;(3)BLZ945@NP NPs;(4)NP/Anti-SIRPα NPs;(5)BLZ945@NP/Anti-SIRPα NPs(以NP NPs为计量方式10 mg/kg)。通过尾静脉注射给药,每两天给药一次,共给药7次。每次注射200 μL。监测治疗期间小鼠的体重变化。以小鼠体重为纵坐标,时间为横坐标,绘制小鼠体重变化曲线图。监测周期结束后,将小鼠颈椎脱臼处死,解剖取出其心、肝、脾、肺、肾等各组织器官,切片,HE染色观察其病理学变化。通过治疗期间小鼠的体重变化曲线及各组织器官切片结果,对BLZ945@NP/Anti-SIRPαNPs的安全性进行评价。
图6为制剂安全性评价结果。从小鼠的体重变化曲线图中可发现治疗期间小鼠体重无明显变化,初步表明该制剂的毒副作用较低。经各制剂组治疗后,观察小鼠各组织的病理切片,制剂组与对照组相比并无显著性差异,表明未发生明显病变。小鼠的体重变化曲线及各组织切片结果均表明该制剂毒副作用较低,安全性良好。
在对实施例实验时,还对其他实施例作了相同实验,均取得了相同或相近似的结果,这里不再一一列举,实验清楚的表明,本发明制备的免疫凝胶内部孔洞均匀,结构致密,呈现多孔纤维状网络结构,BLZ945@NP/Anti-SIRPα NPs呈类球形,外层包覆一层抗体,其均一性良好,具有很好的靶向性能,BLZ945和Anti-SIRPα的联合使用,可发挥协同作用的功效,更大程度地逆转M2型巨噬细胞表型,进而实现对肿瘤免疫抑制性微环境的调控,免疫凝胶在体内具有优异的免疫治疗效果,且毒副作用较低,安全性良好。
需要说明的是,上述仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,任何熟悉本专业的技术人员,在不脱离本发明技术方案范围内,当可利用上述揭示的技术内容作出更动或者修饰为等同变化的等效实施例,均落在本发明的保护范围内。本发明步骤(6)中与纤维蛋白原混合的纳米粒子包括但不限于BLZ945@NP/Anti-SIRPα NPs,还包含可发挥光动力、声动力、光热、免疫等治疗方式的纳米粒子。嵌合在纤维蛋白凝胶孔隙的纳米粒子包含其中一种或多种组合。
本发明制备方法简单、材料易得,效果优异,与现有技术相比,有以下突出的优点:
(1)、以普鲁士蓝作为载体,具有很好的介孔结构,可实现对药物的高效负载;此外,普鲁士蓝本身具有过氧化氢酶活性,可分解机体内的过氧化氢产生氧气,缓解免疫抑制性微环境中的乏氧状态;
(2)、将Anti-SIRPα 修饰于普鲁士蓝纳米粒子表面,既可以起到对巨噬细胞的靶向作用,还能阻断CD47-SIRPα 信号轴;恢复巨噬细胞吞噬及抗原呈递能力的同时,还实现了TAM表型的转换;并且缀合Anti-SIRPα 的纳米颗粒在体内可实现更长的循环时间;
(3)、本发明制备的免疫凝胶以CD47-SIRPα 和MCSF-CSF-1R信号轴为靶标,同时阻断两条信号通路,以期实现协同治疗的效果;
(4)、本发明制备的免疫凝胶在制备防止癌症术后复发药物中的应用,既保证了局部免疫治疗纳米粒子的持续控制释放,又实现了术后创口的修复与愈合,相比于系统性的免疫疗法,该免疫凝胶采用的局部性免疫疗法毒性更低,更经济便利,在临床治疗中具有良好的应用前景,有显著的社会和经济效益。
Claims (9)
1.一种调控肿瘤微环境免疫凝胶的制备方法,其特征在于,包含以下步骤:
(1)合成掺杂NO的普鲁士蓝纳米粒子;
(2)对普鲁士蓝纳米粒子进行氨基化修饰,得氨基化修饰的掺杂NO的普鲁士蓝纳米粒子;
(3)将Anti-SIRPα 修饰于普鲁士蓝纳米粒子表面,得Anti-SIRPα 修饰的普鲁士蓝纳米粒子;
(4)将CSF-1R小分子抑制剂BLZ945负载于普鲁士蓝粒子的孔穴中,得负载BLZ945的普鲁士蓝纳米粒子;
(5)制备胶凝制剂与凝血酶溶液;
所述的胶凝制剂为壳聚糖及其衍生物、透明质酸及其衍生物、藻酸及其衍生物、纤维蛋白及其衍生物或聚链烯基聚醚交联的丙烯酸;
(6)将步骤(4)得到的负载BLZ945的普鲁士蓝纳米粒子与胶凝制剂溶液混合均匀,加入凝血酶溶液,在凝血酶的刺激下即得免疫凝胶。
2.根据权利要求1所述的调控肿瘤微环境免疫凝胶的制备方法,其特征在于,包含以下步骤:
(1)、合成掺杂NO的普鲁士蓝纳米粒子:称取PVP-K30粉末1.2 g,溶于30 mM的K3[Fe(CN)6]溶液中,同时加入等摩尔质量的硝普钠,用浓度12 M的HCl调节PH 为 0,室温下搅拌至溶液变为澄透明的黄色,在80℃条件下避光搅拌12 h,用离心洗涤溶剂对产物进行洗涤离心3次,每次10000 rpm/min条件下离心30 min,随后置于真空干燥箱中干燥12h,得粒径为50 nm的掺杂NO的普鲁士蓝纳米粒子;
所述的离心洗涤溶剂为丙酮、乙醇、超纯水中的一种或按体积任意比混合的两种;
(2)、氨基化修饰:取掺杂NO的普鲁士蓝纳米粒子10 mg与2 mg分子量为1800的氨基化组分置于40 mL超纯水中,磁力搅拌24 h后,置于透析袋中透析24 h,除去未修饰上的、游离的氨基化组分,用超纯水洗涤离心,10000 rpm条件下离心10 min,得氨基化修饰的普鲁士蓝纳米粒子;
所述的氨基化组分为聚醚酰亚胺、聚丙烯胺基盐酸盐、聚酰胺-胺型中的一种或按体积任意比混合的两种;
(3)、将Anti-SIRPα 修饰于普鲁士蓝纳米粒子表面:取氨基化修饰的普鲁士蓝纳米粒子10 mg,加入新鲜配制的活化试剂2 mg/mL的EDC与10 mg/mLNHS溶液,混合均匀后置于25℃摇床中活化1 h,将活化后的产物以9000 rpm离心10 min洗涤3-5次,以除去游离的EDC和NHS分子,得氨基活化的普鲁士蓝纳米粒子,并产物分散于PBS溶液中,加10 μg的SIRPα 抗体至氨基活化的普鲁士蓝纳米粒子中,用移液器轻轻吹打混匀,在4℃条件下孵育过夜,采用6000 rpm转速离心,沉淀分散于PBS溶液中,得SIRPα 抗体修饰的普鲁士蓝纳米粒子;
(4)、负载BLZ945:取经抗体修饰的普鲁士蓝纳米粒子10 mg超声分散于超纯水中,得第一溶液,取10 mg的BLZ945小分子抑制剂溶于DMSO溶液中,得第二溶液,将第二溶液在室温搅拌下加入到第一溶液中,搅拌反应24 h后,置于透析袋中透析24 h,去除游离的BLZ945,用超纯水洗涤离心,10000 rpm条件下离心10 min,得负载BLZ945的普鲁士蓝纳米粒子;
(5)、制备胶凝制剂与凝血酶溶液:将胶凝制剂置于生理盐水溶液中,37℃条件下搅拌10 min,配置终浓度为10 mg/mL的胶凝制剂溶液,将冻干凝血酶置于40 mM氯化钙溶液中,配置浓度为50 U/mL的凝血酶溶液;
(6)、制备免疫凝胶:取步骤(4)中得到的负载BLZ945的普鲁士蓝纳米粒子5 mg,将其分散至10 mg/mL的胶凝制剂溶液中,然后取等体积的凝血酶溶液与之混合,置于37℃恒温箱中孵育1-3 min,即成免疫凝胶。
3.根据权利要求1或2所述的调控肿瘤微环境免疫凝胶的制备方法,其特征在于,包含以下步骤:
(1)、合成掺杂NO的普鲁士蓝纳米粒子:称取PVP-K30粉末1.2 g,溶于30 mM的K3[Fe(CN)6]溶液中,同时加入等摩尔质量的硝普钠,用浓度12 M的HCl调节PH 为 0,室温下搅拌至溶液变为澄透明的黄色,在80℃条件下避光搅拌12 h,分别用乙醇、乙醇与超纯水体积比1:1混合液和丙酮,依次对产物进行洗涤、离心,10000 rpm/min条件下离心30 min,随后置于真空干燥箱中干燥12 h,即得到粒径为50 nm的掺杂NO的普鲁士蓝纳米粒子;
(2)、氨基化修饰:取掺杂NO的普鲁士蓝纳米粒子10 mg与2 mg分子量为1800的聚丙烯胺基盐酸盐置于40 mL超纯水中,磁力搅拌24 h后,置于透析袋(截留分子量8 kDa)中透析24 h,除去未修饰上的、游离的聚丙烯胺基盐酸盐,用超纯水洗涤离心,10000 rpm条件下离心10 min,即得氨基化修饰的普鲁士蓝纳米粒子;
(3)、将Anti-SIRPα 修饰于普鲁士蓝纳米粒子表面:取氨基化修饰的普鲁士蓝纳米粒子10 mg,加入新鲜配制的活化试剂2 mg/mL的EDC与10 mg/mLNHS溶液,混合均匀后置于25℃摇床中活化1 h,将活化后的产物以9000 rpm离心10 min洗涤3次,以除去游离的EDC和NHS分子,得氨基活化的普鲁士蓝纳米粒子,并产物分散于PBS溶液中,加10 μg的SIRPα 抗体至氨基活化的普鲁士蓝纳米粒子中,用移液器轻轻吹打混匀,在4℃条件下孵育过夜,采用6000 rpm转速离心,沉淀分散于PBS溶液中,即得粒径为40-100 nm的Anti-SIRPα 修饰的普鲁士蓝纳米粒子;
(4)、负载BLZ945:取经抗体修饰的普鲁士蓝纳米粒子10 mg超声分散于超纯水中,得第一溶液,取10 mg的BLZ945小分子抑制剂溶于DMSO溶液中,得第二溶液,将第二溶液在室温搅拌下加入到第一溶液中,搅拌反应24 h后,置于透析袋(截留分子量8 kDa)中透析24 h,去除游离的BLZ945,用超纯水洗涤离心后,10000 rpm条件下离心10 min,即得负载BLZ945的普鲁士蓝纳米粒子;
(5)、制备纤维蛋白原溶液与凝血酶溶液:将冻干纤维蛋白原置于生理盐水溶液中,37℃条件下搅拌10 min,配置终浓度为10 mg/mL的纤维蛋白原溶液,将冻干凝血酶置于40 mM氯化钙溶液中,配置终浓度为50 U/mL的凝血酶溶液;
(6)、制得免疫凝胶:取步骤(4)中得到的负载BLZ945的普鲁士蓝纳米粒子5 mg,将其分散至10 mg/mL的纤维蛋白原溶液中,然后取等体积的凝血酶溶液与之混合,置于37℃恒温箱中孵育1-3 min,即得免疫凝胶。
4.根据权利要求1或2所述的调控肿瘤微环境免疫凝胶的制备方法,其特征在于,包含以下步骤:
(1)、合成掺杂NO的普鲁士蓝纳米粒子:称取PVP-K30粉末1.2 g,溶于30 mM的K3[Fe(CN)6]溶液中,同时加入等摩尔质量的硝普钠,用浓度12 M的HCl调节PH 为 0,室温下搅拌至溶液变为澄透明的黄色,在80℃条件下避光搅拌12 h,分别用乙醇、乙醇与超纯水体积比1:1混合液和超纯水,依次对产物进行洗涤、离心,10000 rpm/min条件下离心30 min,随后置于真空干燥箱中干燥12 h,得粒径为50 nm的掺杂NO的普鲁士蓝纳米粒子;
(2)、氨基化修饰:取掺杂NO的普鲁士蓝纳米粒子10 mg与2 mg分子量为1800的聚醚酰亚胺置于40 mL超纯水中,磁力搅拌24 h后,置于透析袋中透析24 h,除去未修饰上的、游离的聚醚酰亚胺,用超纯水洗涤离心,10000 rpm条件下离心10 min,得氨基化修饰的普鲁士蓝纳米粒子;
(3)、将Anti-SIRPα 修饰于普鲁士蓝纳米粒子表面:取氨基化修饰的普鲁士蓝纳米粒子10 mg,加入新鲜配制的活化试剂2 mg/mL的EDC与10 mg/mLNHS溶液,混合均匀后置于25℃摇床中活化1 h,将活化后的产物以9000 rpm离心10 min洗涤4次,以除去游离的EDC和NHS分子,得氨基活化的普鲁士蓝纳米粒子,并将产物分散于PBS溶液中,加入10 μg的SIRPα抗体至氨基活化的普鲁士蓝纳米粒子中,用移液器轻轻吹打混匀,在4℃条件下孵育过夜,采用6000 rpm转速离心,沉淀分散于PBS溶液中,得SIRPα 抗体修饰的普鲁士蓝纳米粒子;
(4)、负载BLZ945:取经抗体修饰的普鲁士蓝纳米粒子10 mg超声分散于超纯水中,得第一溶液,取10 mg 的BLZ945小分子抑制剂溶于DMSO溶液中,得第二溶液,将第二溶液在室温搅拌下加入到第一溶液中,搅拌反应24 h后,置于透析袋中透析24 h,去除游离的BLZ945,用超纯水洗涤离心后,10000 rpm条件下离心10 min,得负载BLZ945的普鲁士蓝纳米粒子;
(5)、制备纤维蛋白原溶液与凝血酶溶液:将冻干纤维蛋白原置于生理盐水溶液中,37℃条件下搅拌10 min,配置终浓度为10 mg/mL的纤维蛋白原溶液,将冻干凝血酶置于40 mM氯化钙溶液中,配置浓度为50 U/mL的凝血酶溶液;
(6)、制备免疫凝胶:取步骤(4)中得到的负载BLZ945的普鲁士蓝纳米粒子5 mg,将其分散至10 mg/mL的纤维蛋白原溶液中,然后取等体积的凝血酶溶液与之混合,置于37℃恒温箱中孵育1-3 min,即成免疫凝胶。
5. 根据权利要求1-4任一项所述的调控肿瘤微环境免疫凝胶的制备方法,其特征在于,所述的步骤(4)所制备的负载BLZ945的普鲁士蓝纳米粒子粒径为40-100 nm。
6.根据权利要求2-4任一项所述的调控肿瘤微环境免疫凝胶的制备方法,其特征在于,所述的步骤(4)所制备的负载BLZ945的普鲁士蓝纳米粒子为注射剂、口服剂或植入剂。
7.根据权利要求2-4任一项所述的调控肿瘤微环境免疫凝胶的制备方法,其特征在于,所述的免疫凝胶为喷雾、注射溶液或涂抹凝胶。
8. 根据权利要求2-4任一项所述的调控肿瘤微环境免疫凝胶的制备方法,其特征在于,所述的步骤(2)和(4)中透析袋的截留分子量为8 kDa。
9.权利要求1-8任一所述方法制备的免疫凝胶在制备防止癌症术后复发药物中的应用。
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